DESCRIPTION: Polymersomes are vesicles prepared from synthetic, amphiphilic-block copolymers. They have several advantages over liposomes, including enhanced stability, longer circulation times, mechanical robustness, and the ability to carry large quantities of hydrophobic and hydrophilic drug molecules. The hydrophilic block of the copolymers is usually polyethyleneglycol (PEG), imparting the long-circulating property to the resultant polymersomes. Because of robustness, the polymersomes require a stimulus to sufficiently disturb the compact bilayer, and to release the encapsulated contents. Although liposomes are extensively studied for drug delivery applications, the potential of polymersomes for targeting, triggered contents release, and simultaneous imaging remains largely unexplored and undeveloped. In this application, we are proposing to synthesize amphiphilic block copolymers, and prepare long-circulating polymersomes. The polymersomes will be actively targeted to tumor tissues, and triggered to release the encapsulated contents upon internalization in the cancer cells. In addition, we will encapsulate gas bubbles in the polymersomes to render them echogenic. This will allow us to perform simultaneous ultrasound imaging, and exert an addition control over contents release employing diagnostic frequency ultrasound. The effectiveness of the proposed multimodal polymersomes will be demonstrated using spheroid cultures and orthotopic mouse model for castration resistant prostate cancer. The Aims of the proposed studies are summarized below. (1) Synthesis of amphiphilic block copolymers, and preparation of echogenic, trigger-releasable polymersomes. We will synthesize bock copolymers containing PEG as the hydrophilic part. The structure as well as the molecular weight of the hydrophobic block will be systematically varied to optimize the polymersome formation process. In order to impart stimuli-responsive property, we will connect the synthesized hydrophilic and hydrophobic polymer blocks via either a reduction-sensitive (for intracellular release) or a hypoxia-sensitive link (for extracellular release). For active targeting and tumor penetration, we will chemically attach the reported cyclic iRGD peptide to the PEG terminus. Echogenic polymersomes will be prepared from these polymers, encapsulating the angiogenesis inhibitor suntinib (for extracellular release) or the anticancer drug docetaxel, and the histone deacetylase inhibitor mocetinostat (for intracellular release). (2) Acoustic characterization of the echogenic polymersomes. We will demonstrate reduction and hypoxia- mediated release of encapsulated contents employing physiologically-relevant concentrations of the cellular reducing agent, glutathione and hypoxic conditions. Echogenicity of the polymersomes will be investigated using detailed acoustic characterization, measuring attenuation, linear, and nonlinear scattering. We will determine the resonance characteristics. We will determine the threshold excitation level for destruction of these polymersomes by measuring time dependent attenuation under progressively higher acoustic excitations. The effect of ultrasound on altering the rate of content release from the polymersomes will be investigated. Optimum acoustic parameters (excitation frequency, pulse waveform and strength) will be determined for linear and nonlinear (harmonic and subharmonic) imaging modalities. (3) Demonstration of effectiveness of the drug delivery system employing spheroid cell cultures and orthotopic mouse model of castration resistant prostate cancer. We will generate the three dimensional spheroids of the castration resistant, prostate cancer cells to evaluate the effectiveness of our polymersomes to deliver the encapsulated drugs. For intracellular delivery, we will probe the synergistic effects of docetaxel and mocetinostat to overcome drug resistance. We will determine the penetration of the polymersomes, cytotoxicity, and reduction in growth of the cancer cells using the tumor spheroids. Subsequently, we will generate orthotopic mouse models of castration resistant prostate cancer to evaluate the effectiveness of our polymersome formulation to deliver their encapsulated drugs. The effect of applied diagnostic frequency ultrasound will be determined, and the spheroids and the tumors in mice will be imaged. We will further determine the effects of the released drugs in reducing the cancer stem cells, the therapeutic regimen for the formulations, remission of the tumors, and potential toxicity of the drugs in different organs.